Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography–triple quadrupole mass spectrometry‐based quantitative protein assays use bottom‐up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top‐down reversed‐phase liquid chromatography–triple quadrupole mass spectrometry method for analysis of cancer‐related intact proteins was evaluated. Seven growth factors (5.5–26.5 kDa; isoelectric points: 4.6–9.9) were analyzed on a wide‐pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1–500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.     

Hydrophilic interaction liquid chromatography--mass spectrometry for the analysis of paralytic shellfish poisoning (PSP) toxins

Hydrophilic interaction liquid chromatography (HILIC) was examined for the separation of paralytic shellfish poisoning (PSP) toxins using the stationary phase TSK-gel Amide-80. The parameters tested included type of organic modifier and percentage in the mobile phase, buffer concentration, pH, flow rate and column temperature. Using mass spectrometric (MS) detection, the HILIC column allowed the determination of all the major PSP toxins in one 30 min analysis with a high degree of selectivity and sensitivity. The high percentage of organic modifier in the mobile phase and the omission of ion pairing reagents, both favored in HILIC, provided limits of detection (LOD) in the range 50-100 nM in selected ion monitoring (SIM) mode on a single quadrupole LC-MS system. LOD in selected reaction monitoring (SRM) mode on a sensitive triple quadrupole system were as low as 5-30 nM. Excellent linearity of response was observed.     

Simultaneous quantitation of nucleosides,nucleobases, amino acids,and alkaloids in mulberry leaf by ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry

An ultra high performance liquid chromatography coupled with a triple quadrupole mass detection and electrospray ionization mass spectrometry method has been established for the simultaneous determination of the 14 nucleosides and nucleobases, 24 amino acids and two main alkaloids in mulberry leaf. In this method, a complicated mobile phase, the flow rate of which was 0.4 mL/min, was applied to the gradient elution, which provided a satisfied separation of the 40 compounds. The present method was validated, and sufficient reproducibility and accuracy were obtained for the quantitative measurement of the 40 compounds. The method was subsequently applied to ten mulberry leaves and the results showed that almost all of these samples were rich in nucleosides, nucleobases, amino acids, and alkaloids. The proposed method, which is convenient and economical, could serve as a prerequisite for the quality control of mulberry leaf herbs and be applied analogously to other Chinese medicines.     

液质联用分析葛根提取物及中药片剂中异黄酮类化合物

采用反相C18毛细管液相色谱柱,以乙腈(含0.1%(体积分数,下同)三氟乙酸)和水(含0.1%三氟乙酸)为流动相梯度洗脱,在26 min内分离了葛根异黄酮提取物以及愈风宁心片中的主要成分。采用毛细管液相色谱/四极杆飞行时间串联质谱仪对葛根提取物以及片剂中的几种主要异黄酮类化合物做了结构分析,发现葛根素是主成分(提取物中其平均质量分数是13.32%;片剂中每片含量19.28~24.34 mg)。对微量未知化合物,用它们的子离子谱图与已知化合物的谱图比较,推测其成分为3′-甲氧基葛根素和3′-甲氧基大豆苷。     

纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。     

Microfabricated refractive index gradient based detector for reversed-phase liquid chromatography with mobile phase gradient elution

Typical refractive index (RI) detectors for liquid chromatography (LC) are not well suited to application with mobile phase gradient elution, due to the difficulty in correcting for the detected baseline shift during the gradient. We report a sensitive, highly reproducible, microfabricated refractive index gradient (micro-RIG) detector that performs well with mobile phase gradient elution LC. Since the micro-RIG signal remains on-scale throughout the mobile phase gradient, one can apply a baseline correction procedure. We demonstrate that by collecting two mobile phase gradient blanks and subtracting one of them from the other, a reproducible, flat baseline is achieved. Therefore, subtracting a blank from a separation provides a baseline corrected chromatogram with reasonably high signal-to-noise ratio for eluting analytes. The micro-RIG detector uses a collimated diode laser beam to optically probe a RIG formed perpendicular to the laminar flow direction within a microfabricated borosilicate glass chip. The chip-based design of the detector is suitable for either traditional bench-top or LC-on-a-chip technologies. We report reversed phase high performance liquid chromatography (RP-HPLC) separations of proteins and polymers, over mobile phase gradient conditions of 67% A:33% B to 3% A:97% B by volume, where A is 96% methanol:3.9% water:0.1% trifluoroacetic acid (TFA), and B is 3.9% methanol:96% water:0.1% TFA. The separations were performed on a Jupiter 5 mu C4 300 A 150 mm x 1.0 mm Phenomenex column at a flow rate of 20 microl/min. Viscosity changes during the mobile phase gradient separation are found to shift the on-chip merge position of the detected concentration gradient (i.e., RIG), in a reproducible fashion. However, this viscosity effect makes detection sensitivity vary throughout the mobile phase gradient, due to moving the optimized position of the probe beam in relation to the analyte concentration gradient being probed. None-the-less, consistent limits of detection (LODs) were achieved. The 3-sigma deflection angle LOD was 16 microrad for micro-RIG detection, corresponding to an injected concentration LOD of 7 ppm (mass/mass) for cytochrome c.     

Simultaneous determination of neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid and geniposide in rat plasma by UPLC‐MS/MS and its application to a pharmacokinetic study after administration of Reduning injection

A simple, specific and sensitive ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method was established and validated for simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and geniposide in rat plasma using puerarin as an internal standard (IS). Plasma samples were pretreated by a one‐step direct protein precipitation procedure with acetonitrile after acidified using as little as 50 μL plasma. Chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 µm) at a flow rate of 0.2 mL/min by a gradient elution, using 0.2% acetic acid–methanol as mobile phase. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with negative ion mode. Calibration curves showed good linearity (r > 0.995) over wide concentration ranges. The intra‐ and inter‐day precisions were <15%, and the accuracy was within ±8.0%. The validated method was successfully applied to a pharmacokinetic study of the four bioactive components in rats after intravenous administration of Reduning injection. Copyright © 2014 John Wiley & Sons, Ltd.     

Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.     

Simultaneous ultra‐performance liquid chromatography–tandem mass spectrometry determination of six components in rat plasma after oral administration of Smilacis glabrae Roxb. extract

In this study, an accurate and reliable method of ultra‐performance liquid chromatography coupled with a triple‐quadrupole tandem mass spectrometry was firstly developed and fully validated for the simultaneous determination of epicatechin, neoastilbin, astilbin, isoastilbin, engeletin and resveratrol in rat plasma after administration of Smilacis glabrae Roxb. extract. Naringenin was used as an internal standard (IS). The analyte and IS were separated on a C₁₈ column by gradient elution with a mobile phase of acetonitrile–0.3% acetic acid at a flow rate of 0.25 mL/min for a total run time of 8 min. The method was validated in terms of selectivity, linearity, precision, accuracy, extraction recovery, matrix effect and stability. The developed method was successfully applied to determine the main pharmacokinetic parameters of six components in rat plasma.     

Determination of teniposide in rat plasma by ultra performance liquid chromatography electrospray ionization tandem mass spectrometry after intravenous administration

A novel, specific and rapid ultra performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for determination of teniposide in rat plasma. A one‐step liquid–liquid extraction method was used and the separation was carried out on an Acquity UPLC^TM BEH C₁₈ column with gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.20 mL/min. A triple quadrupole tandem mass spectrometer in multiple‐reaction monitoring mode via an electrospray ionization interface was used for the detection of teniposide. The detection was complete within 3.0 min. A linear calibration curve was obtained over the concentration range 10–10,000 ng/mL for teniposide, with a lower limit of quantification of 10 ng/mL. The intra‐day precision and inter‐day precision (relative standard deviation) were less than 10.23 and 13.09%, respectively. The developed method was applied for the first time to the pharmacokinetic study of teniposide in rats following a single intravenous administration of 4.5 mg/kg teniposide. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantioselective separation and pharmacokinetic dissipation of cyflumetofen in field soil by ultra‐performance convergence chromatography with tandem mass spectrometry

Little data on the enantioselective separation of cyflumetofen exists, despite the fact that such data are essential to the assessment of the fate and potential toxic effects of cyflumetofen enantiomers. To address this issue, a simple and sensitive method for the enantioselective determination of cyflumetofen enantiomers in soil has been established using ultra performance convergence chromatography tandem triple quadrupole mass spectrometry. The effects of the chiral stationary phases, mobile phase, auto backpressure regulator pressure, column temperature, flow rate of the mobile phase, and compensation pump solvent were evaluated. The proposed method was applied to the study of the pharmacokinetic dissipation of cyflumetofen stereoisomers in soil under greenhouse conditions. The estimated half‐life of cyflumetofen isomers ranged from 12.2 to 13.6 days, and statistically significant enantioselective degradation was observed. This study not only demonstrates that there is an efficient and sensitive method for cyflumetofen enantioseparation, but also provides the first experimental evidence of the pharmacokinetic dissipation of cyflumetofen stereoisomers in the environment.     

Comparison of the separation of proteins of wide-ranging molecular weight via trilobal polypropylene capillary-channeled polymer fiber,commercial superficiously porous,and commercial size exclusion columns

Reversed phase and size-exclusion chromatography methods are commonly used for protein separations, although they are based on distinctly different principles. Reversed phase methods yield hydrophobicity-based (loosely-termed) separation of proteins on porous supports, but tend to be limited to proteins with modest molecular weights based on mass transfer limitations. Alternatively, size-exclusion provides complementary benefits in the separation of higher mass proteins based on entropic, not enthalpic, processes, but tend to yield limited peak capacities. In this study, microbore columns packed with a novel trilobal polypropylene capillary-channeled polymer fiber were used in a reversed phase modality for the separation of polypeptides and proteins of molecular weights ranging from 1.4 to 660 kDa. Chromatographic parameters including gradient times, flow rates, and trifluoroacetic acid concentrations in the mobile phase were optimized to maximize resolution and throughput. Following optimization, the performance of the trilobal fiber column was compared to two commercial-sourced columns, a superficially porous C4-derivatized silica and size exclusion, both of which are sold specifically for protein separations and operated according to the manufacturer-specified conditions. In comparison to the commercial columns, the fiber-based column yielded better separation performance across the entirety of the suite, at much lower cost and shorter separation times.     

An integrated multipath liquid chromatography–mass spectrometry system for the simultaneous preparation,separation, and detection of proteins and small molecules

A multipath liquid chromatography with mass spectrometry instrument was constructed with the help of restricted access media to online segregate small and large molecules. This liquid chromatography system was custom built with five pumps and three two‐position six‐port valves to control the flow in a multipath system for the simultaneous analysis of small molecules and proteins. On separate chromatographic channels, small molecules trapped and proteins excluded from the online restricted access media were analyzed downstream using high‐efficiency columns and a triple quadrupole mass spectrometer. A model sample, which included five proteins and 22 small molecules with different physicochemical properties, was used to evaluate the system. Following injection, the complete multipath separation and detection was performed in 22 min. Protein exclusion by the restricted access media was not quantitative. Four commercial trap columns were evaluated for their exclusion efficiency toward the proteins. Exclusion efficiency varied from <50% to only a maximum of 75% exclusion across the trap columns tested. An attempt was made to optimize the exclusion efficiency using different flow rates, flow rate gradients, and different additives both in the sample and the mobile phases. Protein exclusion was still erratic and generally nonquantitative.     

Quantitative analysis of the farnesyl transferase inhibitor lonafarnib (Sarasartrade mark, SCH66336) in human plasma using high-performance liquid chromatography coupled with tandem mass spectrometry

Lonafarnib is a novel anticancer drug that inhibits farnesyl transferase. To assess its pharmacokinetic properties, we developed a sensitive and quantitative assay using liquid chromatography coupled with tandem mass spectrometry for the determination of lonafarnib levels in human plasma. Sample pretreatment consisted of the addition of an isotopically labeled internal standard and protein precipitation with acetonitrile using 100 microL plasma. Chromatographic separation was performed on an Inertsil ODS-3 analytical column (50 x 2.1 mm i.d., particle size 5 microm) with acetonitrile/water/formic acid (50:50:0.05, v/v/v) as the mobile phase, at a flow rate of 0.2 mL/min. The analytical run time was 8 min. An API365 triple quadrupole mass spectrometer was used for specific and sensitive detection. It was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated using a concentration range of 2.5 to 2500 ng/mL lonafarnib. Validation of the assay was performed according to the most recent FDA guidelines for bioanalytical method validation and all results were within the requirements. The described method was successfully applied to support a clinical phase I trial with lonafarnib.     

Simultaneous quantitation and comparison of eight components in Jiao‐ai decoction and Si‐wu decoction by ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry

An ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry method has been established to evaluate the variations of multiple components of Chinese herbal preparations, Jiao‐ai decoction and Si‐wu decoction, through the simultaneous determination of eight major active compounds with a huge difference in the level of content. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) with a mobile phase consisting of acetonitrile (0.1% formic acid) and water (0.1% formic acid) under gradient elution. A triple quadrupole tandem mass spectrometer was operated in positive and negative ionization modes, respectively, with multiple reaction monitoring for the detection of the eight compounds. All calibration curves showed excellent linear regressions (r > 0.99) within the test range. The precision, repeatability, and stability of the eight compounds were below 5.0% in terms of relative standard deviation. The recoveries were 97.0–102.4% with a relative standard deviation of 1.21–3.65% for all samples. In conclusion, a rapid, sensitive, precise, accurate, and reliable method has been developed for the simultaneous detection of eight active compounds in the pharmaceutical samples of Jiao‐ai decoction and Si‐wu decoction, which can be applied for the multicomponent comparison and further quality control.     

Determination of hydroxysafflor yellow A in biological fluids of patients with traumatic brain injury by UPLC‐ESI‐MS/MS after injection of Xuebijing

A simple, novel, specific, rapid and reproducible ultra‐performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for the determination of hydroxysafflor yellow A (HSYA) in biological fluids (plasma, urine and cerebrospinal fluid) of patients with traumatic brain injury after intravenous injection of Xuebijing (XBJ). Liquid–liquid extraction was performed, and separation was carried out on an Acquity UPLC? BEH C₁₈ column, with gradient elution using a mobile phase composed of methanol and 0.1% formic acid at a flow rate of 0.3 mL/min. A triple quadrupole tandem mass spectrometer with electrospray ionization was used for the detection of HSYA. The mass transition followed was m/z 611.0 → 491. The retention time was less than 3.0 min. The calibration curve was linear in the concentration range from 2 to 6125 ng/mL for cerebrospinal fluid, plasma and urine. The intra‐ and inter‐day precisions were <10%, and the relative standard deviation of recovery was <15% for HSYA in biological matrices. The method was successfully applied for the first time to quantify HSYA in the biological fluids (especially in cerebrospinal fluid) of patients with traumatic brain injury following intravenous administration of XBJ. Copyright © 2014 John Wiley & Sons, Ltd.     

Fast ultra‐high‐performance liquid chromatography with diode array and mass spectrometry method for determination of tadalafil drug substance and its impurities

Tadalafil is used for the treatment of erectile dysfunction. Its related patents expired in 2016, and so related generic drug production is predicted to be increased. This work is focused on developing a fast ultra‐high‐performance liquid chromatography with diode array detection and/or mass spectrometry detection for the separation and determination of tadalafil and its impurities in pharmaceutical samples. A modern reversed‐phase stationary phase with sub‐2 μm particle size, Zorbax StableBond Rapid Resolution High Definition with octylsilane chemically bonded phase to totally porous silica particles, was used for the solving this problem. Column temperature was set at 40 ± 0.1°C. A mobile phase consisting of acetonitrile and aqueous solution of 0.1% (v /v) trifluoroacetic acid for diode array detection detection and 0.05% (v /v) formic acid, both running at a flow rate of 0.62 mL/min, were used to achieve the required separation of all components within a 5 min run. The limit of detection was 3.5 μg/L and the limit of quantification was 10.0 μg/L for the method for both UV and MS detectors. Accurate mass spectra of tadalafil's related impurities are shown for advanced confirmation. The method is directly transferable to routine analysis of tadalafil in pharmaceutical and control laboratories.     

Continuous-flow fast atom bombardment mass spectrometry of oligonucleotides

Although frit-fast atom bombardment (frit-FAB) and continuous-flow FAB mass spectrometry have become standard methods for the analysis of peptides and peptide mixtures, these techniques have not been applied previously to the analysis of oligonucleotides. Mobilephase composition, flow rate, and sample size were optimized for the analysis of oligonucleotides by negative ion frit-FAB mass spectrometry (a type of continuous-flow FAB mass spectrometry). With a mobile phase consisting of methanol/water/triethanolamine (80:20:0.5, v/v/w), flow injection frit-FAB analysis of oligonucleotides showed lower limits of detection compared to standard probe FAB mass spectrometry. For example, in order to obtain a signal-to-noise ratio of 3:1, 38 prnol of d(GTIAAC) were required for frit-FAB mass spectrometry and 62 pmol were required for standard probe FAB mass spectrometry. The largest difference between frit-FAB and standard probe FAB was observed for d(pC)₅, for which the limit of detection by frit-FAB was approximately 11-fold lower than by standard FAB mass spectrometry. Adjustment of the mobile phase to pH 7 with trifluoroacetic acid increased the limit of detection (reduced sensitivity) a minimum of sixfold. Equimolar mixtures of two or three oligonucleotides produced deprotonated molecules in identical relative abundances whether analyzed by frit-FAB or standard probe FAB mass spectrometry. Finally, frit-FAB liquid chromatography mass spectrometry was demonstrated by separating mixtures of oligonucleotides on a β -cyclodextrin high-performance liquid chromatography column with a mobile phase containing methanol, water, and triethanolamine.     

高效液相色谱法测定茶叶中的茶氨酸

建立了未衍生化高效液相色谱法(HPLC)测定茶叶中茶氨酸含量的方法。采用的色谱条件为:C 18 色谱柱,以0.05%(体积分数)三氟乙酸水溶液为流动相,流速1 mL/min ,进样量10 μL,检测波长203 nm。茶氨酸质量浓度在0.02～1 g/L 内,其浓度与峰面积呈良好的线性关系,最低检出限为1.75 ng(S/N=3),回收率为97.2%,相对标准偏差(RSD)为1.7%。同时以高效液相色谱-电喷雾离子化质谱对所分离的茶氨酸进行了纯度鉴定。方法具有精确、灵敏、流动相组成简单等特点。     

Separation and determination of polyurethane amine catalysts in polyether polyols by using UHPLC–Q‐TOF‐MS on a reversed‐phase/cation‐exchange mixed‐mode column

A simple, selective, and accurate ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established and validated for the efficient separation and quantification of polyurethane amine catalysts in polyether polyols. Amine catalysts were primarily separated in polyether polyol‐based sample by solid‐phase extraction, and further baseline separated on a reversed‐phase/cation‐exchange mixed‐mode column (SiELC Primesep™ 200) using 0.1% trifluoroacetic acid/acetonitrile as a mobile phase in gradient elution mode at a flow rate of 0.2 mL/min. High‐resolution quadrupole time‐of‐flight mass spectrometry analysis in electrospray ionization positive mode allowed the identification as N,N′‐bis[3‐(dimethylamino)propyl]urea, N‐[2‐(2‐dimethylaminoethoxy)ethyl]‐N‐methyl‐1,3‐propanediamine, and N,N,N′,N′‐tetramethyldipropylenetriamine. The method was validated and presented good linearity for all the analytes in blank matrices within the concentration range of 0.20–5.0 or 0.1–2.0 μg/mL with the correlation coefficients (R²) ranging from 0.986 to 0.997. Method recovery ranged within 81–105% at all three levels (80, 100, and 120% of the original amount) with relative standard deviations of 1.0–6.2%. The limits of detection were in the range of 0.007–0.051 μg/mL. Good precision was obtained with relative standard deviation below 3.2 and 0.72% for peak area and retention time of three amines, respectively.